Summary: Our laboratory studies signal transduction in the immune system involving tyrosine phosphorylation and non-receptor tyrosine kinases, molecules required for intracellular signaling pathways involved in normal cellular growth and differentiation as well as the abnormal growth and development involved in cancer. Mutations affecting such signaling pathways have been found to give rise to multiple human disorders including several primary immunodeficiencies. Using a combination of genetics, protein biochemistry and cell biology, our goals are to understand how these molecules contribute to normal function of cells in the immune system. Our goals include understanding how manipulation of these pathways can help in the understanding of diseases of the immune system and in the development of therapeutics for disease. A large portion of our work is focused on studies of the Tec family of tyrosine kinases. Mutations affecting the prototypical member of this family, Btk, are responsible for the human genetic disorder X-linked agammmaglobulimemia. Btk is required for normal B cell development and function. We have shown that mutation of Tec family kinases expressed in T cells can also severely impair T lymphocyte function in mice and influence responses to infections in vivo. Previous studies have demonstrated that the Tec kinases are critical for antigen receptor induced activation of phospholipase-c gamma, a key enzyme required for Ca++ mobilization. Over the last two years, we have described and studies a newly discovered role forTec kinases in regulation of the actin cytoskeleton in T lymphocytes. We have recently reported altered activation of Cdc42, a Rho family GTPase that is an upstream activator of the Wiskott Aldrich Syndrome Protein (WASp) in cells deficient for the Tec kinase Itk. and altered subcellular localization of the guanine nucleotide exchange factor Vav. Mutations affecting both WASp and Vav have been implicated in primary immunodeficiencies. Using genetic and biochemical methods, we have recently extended our findings to help determine the interactions between Itk and these key regulators of the T cell actin cytoskeleton organization and have demonstrated a key role for Tec kinases in activated of lymphocyte adhesion. Our results place the Tec kinases as critical regulators of the actin cytoskeleton, cell adhesion and migration and suggest that cytoskeletal defects may contribute to the phenotypes associated with Tec kinase deficiency including those seen in X-linked agammaglobulinemia. In continuing work, we are also examining the effects of mutation of Tec kinases on T cell responses in vivo. We had previously found that mutation of the Tec kinases alters the balance of T helper cell differentiation and cytokine production. We are now extending these studies using a combination of transgenic and gene-targeted mice to alter expression of the Tec kinases in T lymphocytes. Additionally, we are examining the role of related signaling molecules including WASp in T helper cell differentiation and in vivo responses to infection. As an extension of these studies, we are examining other signaling molecules involved in T helper cell differentiation including SAP, a small SH2 containing adaptor protein, mutations of which are associated with the genetic disorder X-linked proliferative syndrome (XLP). SAP binds to and helps recruit the tyrosine kinase Fyn to the intracellular tails of SLAM and related co-stimulatory receptors. We had previously generated mice deficient in SAP and have found that upon challenge with infectious agents, these mice recapitulated features of XLP, including increased T cell activation and decreased antibody production. In the last year, we have extended these findings to demonstrate a critical role of SAP in responses to immunization. We further showed that the impaired antibody responses in these mice is secondary to a defect in CD4+ T cells, ie SAP deficient T cells fail to provide an essential signal to B cells for generating long-term antibody responses, a critical step for the development of successful immunization and immune responses, the hallmark of successful vaccine development. Understanding the cellular interactions and signals that are defective in these mice is therefore of high importance or understanding the requirements for successful vaccine development. To understand the defect in T cells in the SAP-deficient mice, we have continued to examine T cell function and biochemistry of T cell activation in cells from SAP-deficient mice. We have found that T cells from SAP deficient mice show increased Th1 cytokine production (IFN-g) and dramatic defects in Th2 cytokine production (IL-4, 5, and 10) in response to TCR stimulation. We have further demonstrated that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production, but is secondary to decreased TCR-induced PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation and nuclear NF-kappaB1/p50 levels. Re-expression of wildtype (WT), but not a Fyn-binding mutant of SAP rescued the defects in both PKC-theta recruitment and IL-4 production in SAP-deficient cells, demonstrating that this phenotype requires SAP-Fyn recruitment. Moreover, SLAM engagement increased TCR-mediated PKC-theta recruitment, nuclear p50 levels and IL-4 production in WT but not SAP-deficient T cells, suggesting a potential new role for SLAM in T cell signaling. Our data indicates that a SAP/Fyn pathway is required for the efficient recruitment of PKC-theta/Bcl-10 as well as proper patterns of activation of NF-kappaB, and suggests a potentially novel pathway of T helper 2 (IL-4) cytokine regulation. Nonetheless, recednt studies suggest that the humoral defects may involve distinct signaling pathways. These findings provide a molecular framework for probing T cell function and immune cell dysregulation in this complex disorder.